ABSTRACT The manipulation of gene expression in Mycobacterium tuberculosis (Mtb) remains a challenge. In particular the regulation of multiple genes simultaneously is difficult and prohibitively time consuming. The CRISPR-Cas system is an RNA-guided bacterial adaptive immune system with transformative potential for gene editing, and CRISPR interference (CRISPRi), is a new tool to control gene expression in many organisms. CRISPR systems are inherently multi-gene regulatory systems, based on their ability to express and process a CRISPR array into many dozen RNAs that can each target a different gene. This unique multi-gene targeting function has not yet been exploited because not all required precursor RNA processing components are known for most systems, precluding their heterologous expression. However, these limitations apply only to the orthogonal use of CRISPR systems when heterologous expression of all components is required. Endogenous CRISPR systems, in contrast, provide all necessary components for processing and interference and are optimally tuned to function in their particular host. Here, we will repurpose the endogenous Mtb type III CRISPR system for the regulation of genes in any strain and for multi-gene regulation.